1. Field of the Invention.
The crystalline cephalosporin hydrate of the present invention possesses in general the usual attributes of that family of antibacterial agents and is particularly useful in the treatment of bacterial infections by both oral and parenteral administration.
2. Description of the Prior Art.
The literature concerning this class of antibacterial agents has been reviewed frequently; two recent reviews are The Cephalosporins Microbiological, Chemical and Pharmacological Properties and Use in Chemotherapy of Infection, L. Weinstein and K. Kaplan, Annals of Internal Medicine, 72, 729-739 (1970) and Structure Activity Relationships Among Semisynthetic Cephalosporins, M. L. Sassiver and A. Lewis, Advances in Applied Microbiology, edited by D. Perlman, 13, 163-236 (1970), Academic Press, New York. Additional reviews which pay particular attention to the patent literature are found in U.S. Pat. Nos. 3,776,907, 3,776,175 and 3,759,904. Solvates, and hydrates in particular, are often encountered in the cephalosporin field, e.g. U.S. Pat. Nos. 3,280,118, 3,502,663, 3,655,656, 3,692,781, 3,708,478 and 3,714,157.
7-[D-.alpha.-Amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5-y lthiomethyl)-3-cephem-4-carboxylic acid is a new cephalosporin, also called BL-S640, which is described and claimed by my colleagues David Willner and Leonard B. Crast, Jr. in U.S. application Ser. No. 318,340 filed Dec. 26, 1972; the entire disclosure of that application is incorporated herein by reference.
In the preparation of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid by the procedures disclosed in the above-mentioned U.S. applicatian Ser. No. 318,340, the crude amorphous product obtained in chemical production is rather heavily contaminated with residues of the reagents and with various decomposition products from which it can not be separated in reasonable yield by recrystallization or the other usual techniques such as washing with solvents. All efforts to purify the amorphous crude product by forming a crystalline zwitterion or a hydrate thereof by conventional methods, e.g. crystallization from an aqueous reaction mixture by adjustment of the pH to the isoelectric point, have failed. The problem of purifying this particular cephalosporin is further complicated by the fact that at alkaline pH, i.e. pH 7.0 or higher, the compound degrades very rapidly by loss of the thiol moiety.
In an attempt to isolate and purify the amorphous 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid, the amorphous crude product has been successfully converted into a crystalline methanol solvate. This methanolate, however, is undesirable for human pharmaceutical use because of the toxicity of methanol. In addition the methanol solvate provides little purification of the crude product as measured by any increase in biopotency, decrease in color or reduction in impurity content. An ethanol solvate has also been prepared and, while crystalline, its formation was not accompanied by any significant purification. When suspended in water, moreover, the ethanolate gradually lost its ethanol to change into a solid tacky form which lost crystallinity, did not suspend evenly and gummed. No way was found to remove the solvents from the solvates in order to obtain essentially anhydrous pure compound and the products so obtained became tacky and were not useful for pharmaceutical formulations. Lyophilization of water solutions of the methanol or ethanol solvates (5 - 10 mg./ml.) gave an amorphous monohydrate product which was biologically unstable (lost 37% of its potency when stored at 56.degree. C. for 1 month) and unacceptable for pharmaceutical use.
It was an objective of the present invention to provide a stable, nontoxic, non-solvated crystalline hydrate of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid which would be substantially free of impurities and useful in preparing pharmaceutical dosage forms of the cephalosporin antibiotic for both oral and parenteral administration.
The present invention provides a novel crystalline sesquihydrate of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid and processes for its preparation. The hydrate of the present invention is a particularly useful form of the above-mentioned cephalosporin in that it is produced substantially free of the impurities found in samples of the cephalosporin made by practical commercial processes. In addition, it possesses good biological stability (as a solid it loses less than 6% of its bioactivity when stored for 1 month at 56.degree. C.) and the desirable physical properties needed for formulation into both oral and parenteral dosage forms. When suspended in water the crystalline sesquihydrate does not lose biological activity and does not undergo deleterious changes on standing such as loss of crystallinity, uneven suspension, oiling, clumping, settling or tackiness. The crystalline sesquihydrate is a useful broad-spectrum antibacterial agent and is found to provide effective blood levels on both oral and parenteral administration.
The novel crystalline form of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid sesquihydrate described and claimed herein exhibits essentially the following X-ray powder diffraction properties:
Line Spacing d(A) Relative Intensity ______________________________________ 1 9.31 45 2 6.77 93 3 6.17 32 4 5.56 14 5 4.42 100 6 3.97 76 7 3.78 95 8 3.55 55 9 3.41 53 10 3.10 26 11 3.01 19 12 2.94 9 13 2.83 17 14 2.77 23 15 2.55 19 16 2.32 10 17 2.23 12 18 2.05 10 19 1.97 6 20 1.88 7 ______________________________________
The details for this determination of X-ray diffraction properties are as follows:
A small amount of sample was sealed in a 0.2 mm. diameter low scattering glass capillary tube which was mounted for exposure in a 114.6 mm. diameter Debye-Scherrer powder diffraction camera. The exposure time was 8 hours on a Norelco X-ray Generator operated at 35 KV-20 mA using a standard focus copper target X-ray tube (weighted CuK.sub..alpha. wavelength .lambda.= 1.5418 A). Kodak No-Screen X-Ray Film was used and developed for 3 minutes at 20.degree. C. in Kodak Liquid X-ray Developer.
A very small amount of crystalline sodium fluoride was mixed in with some samples to provide internal calibration. In addition, a sample of pure NaF was run through the complete procedure for the same purpose.
The films were read on a Norelco Debye-Scherrer film reader, recording the positions of the diffraction rings to the nearest 0.05 mm. The data were corrected for film shrinkage and the interplanar spacings ( d -spacings) were calculated from the corrected data. A computer program (XRAY, by P. Zugenmaier) was used for all calculations. The accuracy in the resulting d-spacing data was .about. 1%.
An intensity record of all films was obtained using a Joyce-Loeble Mark IIIC Recording Microdensitometer (scan ratio 5:1, 0.1 O.D. wedge). Relative intensities on a scale 1-100 were assigned to all recognizable diffraction rings using peak intensities corrected for the background reading.
A sample of the crystalline sesquihydrate product was subjected to IR and NMR analyses and the functional group data from the spectra are summarized below:
IR (as KBr disc) ______________________________________ 2400 - 3600 cm.sup..sup.-1 (broad overlapping peaks) amide NH, NH.sub.3 .sup.+, OH(H.sub.2 O) 1780 .beta.-lactam C=O 1707 amide C=O 1570 COO.sup.- 1520 aromatic C=C NMR (DMSO, dilute DCl) ______________________________________ 7.96 ppm .gamma. singlet, 1H, H.sub.a 6.7 - 7.6 multiplet, 4H, H.sub.b 5.7 doublet, 1H, H.sub.c 4.9 - 5.2 multiplet, 2H, H.sub.d, H.sub.e 3.2 - 4.2 multiplet, 4H, H.sub.g, H.sub.f 1.1 doublet* ______________________________________ * residual propylene glycol. ##SPC1##
The crystalline sesquihydrate has a decomposition range of 188.degree.-194.degree. C. and, when observed under a polarizing microscope, appears as micro crystals. The sesquihydrate has a solubility in water of approximately 10 mg./ml. at 25.degree. C.
The present invention further provides a process for the preparation of crystalline 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid sesquihydrate; which process comprises suspending 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid methanol solvate in water at a pH range of approximately 2.5 - 7.0 and recovering the desired crystalline sesquihydrate product from the aqueous reaction mixture.
A preferred embodiment of the present invention is a process for the preparation of crystalline 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid sesquihydrate; which process comprises treating an aqueous suspension of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid methanol solvate with sufficient base to raise the pH of the aqueous suspension to between about 6 and 7 and preferably in the range of about 6.2 - 6.7 at a temperature in the range of about 5.degree. C. to 85.degree. C. for a period of time of from about 1 to 24 hours; recovering the crystalline hydrate from the aqueous reaction mixture and air drying at a temperature between about 37.degree.-45.degree.C. to produce the desired crystalline sesquihydrate product.
The methanol solvate of 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid is slurried in water to form an aqueous suspension having a pH in the range of about 3-4. The methanol solvate starting material may be prepared directly from the crude amorphous cephalosporin zwitterion by the procedures described in U.S. application Ser. No. 318,340, e.g. crystallization from a methanol solution of the crude zwitterion, or by the improved procedures described below in the section entitled "Preparation of the Starting Materials". Most advantageously, however, the crude cephalosporin zwitterion is recovered as the methanol solvate, the methanol solvate is converted to a propylene glycol solvate and the propylene glycol solvate finally reconverted to a purer crystalline methanol solvate which is employed as starting material in the process of the present invention for preparation of the crystalline sesquihydrate. The procedures for preparing the methanol and propylene glycol solvates are described below in the "Preparation of the Starting Materials" section. The above-mentioned preferred purification procedure minimizes the impurities found in the methanolate starting material and results in production of the highest quality hydrate product. Since water does not appear to replace propylene glycol residues in the crystalline methanolate, it is also desirable to use methanol solvate which is substantially free of propylene glycol impurities. This propylene glycol-free methanol solvate may be prepared for example by recrystallizing the methanolate from methanol until substantially all of the propylene glycol impurities have been removed. The aqueous suspension of methanol solvate may be of any desired concentration, but it is preferred for highest yields to employ a concentration of about 250 mg. methanolate per ml. water. The methanolate is preferably ground to a particle size of less than 200 mesh.
After preparing the aqueous suspension of methanol solvate and preferably stirring for a period of time of from about 1 to 24 hours, the crystalline hydrate may be recovered from the aqueous reaction mixture by conventional methods such as filtration or centrifugation. The product is washed with water and air dried at a temperature of between about 37.degree.C. and 45.degree.C. preferably for a period of from about 24 to 48 hours.
In the preferred method the methanol solvate is slurried in water to form a aqueous suspension and the pH of the suspension is then slowly raised to a value such that the maximum amount of crystalline hydrate crystallizes out of the reaction mixture. Best results are obtained when the pH is raised by addition of base to about 6-7 and most preferably in the range of about 6.2 - 6.7. The nature of the base used to effect the pH change is not important and any water-miscible base is acceptable. The most preferred bases because of availability and cost are the alkali metal hydroxides, e.g. sodium or potassium hydroxide. The base is added slowly with stirring for a time sufficient to cause precipitation of the desired crystalline hydrate. The formation of the crystalline hydrate is believed to occur in a short time but it is preferred to stir the reaction mixture for a period of from about 1 to 24 hours, most preferably about 2 hours, so as to permit complete reaction and formation of good quality crystals. The temperature of the reaction mixture during the pH adjustment step is not particularly critical and may advantageously be in the range of from about 5.degree. C. to 85.degree. C. Most conveniently, however, the entire process is carried out at room temperature.
The crystalline sesquihydrate is recovered from the reaction mixture by conventional methods such as filtration or centrifugation, washed with water and air dried at temperatures in the range of about 37.degree. - 45.degree. C. preferably for a period of time of from about 24-48 hours.
A slight variation of the above preferred process comprises adding the methanolate starting material directly to an aqueous solution having a pH of about 6-7 and most preferably in the range of about 6.2 - 6.7. The aqueous methanolate suspension is then slurried preferably for between about 1 to 24 hours and the crystalline hydrate recovered as described above.
Upon recovery of the crystalline hydrate from the reaction mixture and cooling to room temperature, the hydrate is ordinarily isolated in the form of the crystalline dihydrate. The dihydrate is found to lose one-half molecule of bound water under very mild conditions, however, and thus air drying of the product hydrate at a temperature in the range of about 37.degree. - 45.degree. C. for 24 hours will convert the dihydrate to the more stable sesquihydrate crystalline product. Air drying of the crystalline dihydrate at room temperature for a period of about 24 hours in the presence of dry laboratory air has also been found to result in conversion to the preferred crystalline sesquihydrate form. The crystalline sesquihydrate of the present invention may be subjected to more extreme drying conditions, i.e. air drying at temperatures above about 45.degree. C. and/or vacuum drying to form other crystalline hydrates having lesser amounts of bound water. Thus, the crystalline sesquihydrate having 1.5 moles of water per mole of cephalosporin zwitterion can be converted to a crystalline monohydrate by air drying at 56.degree. C. for 24 hours. Upon vacuum drying at 56.degree. C. for 24 hours a crystalline hemihydrate is formed having 0.5 moles of bound water per mole of cephalosporin. All of the above-mentioned novel crystalline hydrates are included within the scope of the present invention.
When tested in vitro and in vivo, the crystalline 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)acetamido]-3-(1,2,3-triazol-5- ylthiomethyl)-3-cephem-4-carboxylic acid sesquihydrate of this invention exhibits substantially the same potency and spectrum of activity reported in U.S. application Ser. No. 318,340.
In the treatment of bacterial infections in man, the crystalline 7-[D-.alpha.-amino-.alpha.-(p-hydroxyphenyl)-acetamido]-3-(1,2,3-triazol-5 -ylthiomethyl)-3-cephem-4-carboxylic acid sesquihydrate is administered either orally or parenterally, as preferred by the physician, in an amount of from about 5 to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divided dosage, e.g. three or four times a day. It is administered in dosage units containing, for example, 125, 250 or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units may be in solid form such as tablets or capsules or liquid form such as aqueous solutions or suspensions.